Clonogenic Assay for Radiation Sensitivity

After 48 h of transfection, cells were transferred to complete medium and seeded on 6-well plates in triplicate and exposed to radiation at the doses indicated using a 6-MV X-ray generated by a linear accelerator (Varian 2300EX at a dose rate of 5 Gy/min; Varian MedicalSystems, Palo Alto, CA). After incubation at 37 °C for 14 days, cells were fixed in 100% methanol and stained with 1% crystal violet. Colonies containing >50 cells were counted under a light microscope (Olympus, Japan). The surviving fraction was calculated as previously described^{12 (link)}. Each assay was repeated in three independent experiments.